The present invention relates to compositions and methods for the prevention and treatment of cell proliferative disorders wherein a protein tyrosine kinase or protein tyrosine phosphatase capable of complexing with a member of the SH2- and/or SH3-containing family of adaptor proteins is involved. This invention is based, in part, on the surprising discovery that the adaptor protein, GRB-2, binds the intracellular BCR-ABL tyrosine kinase product in vivo and is necessary for the activation of the oncogenic potential of the BCR-ABL product and that disruption of the signaling capability of GRB-2 can reverse the transformed phenotype of cells and reduce tumor growth in animals. The present invention further relates to protein tyrosine kinase/adaptor protein complexes and the uses of these complexes for the identification of agents capable of disrupting the interaction between the members of such complexes.
2.1. Protein Phosphorylation and Signal Transduction
Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their immediate environment. These extracellular signals are essential for the correct-regulation of such diverse cellular processes as differentiation, contractility, secretion, cell division, contact inhibition, and metabolism. The extracellular molecules, which can include, for example, hormones, growth factors, lymphokines, or neurotransmitters, act as ligands that bind specific cell surface receptors. The binding of these ligands to their receptors triggers a cascade of reactions that brings about both the amplification of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above. In addition to normal cellular processes, receptors and their extracellular ligands may be involved in abnormal or potentially deleterious processes such as virus-receptor interaction, inflammation, and cellular transformation to a cancerous state.
A central feature of this process, referred to as signal transduction (for recent reviews, see Posada et al., 1992, Mol. Biol. Cell 3:583-592; Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255), is the reversible phosphorylation of certain proteins.
The phosphorylation or dephosphorylation of amino acid residues triggers conformational changes in regulated proteins that alter their biological properties. Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classified according to the amino acid residues they act on, with one class being serine-threonine kinases and phosphatases (reviewed in Scott et al., 1992, 2:289-295), which act on serine and threonine residues, and the other class being the tyrosine kinases and phosphatases (reviewed in Fischer et al., 1991, Science 253:401-406; Schlessinger et al., 1992, Neuron 9:383-391; Ullrich et al., 1990, Cell 61:203-212), which act on tyrosine residues. Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative. activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions.
While the majority of protein phosphorylation occurs at serine and threonine amino acid residues, phosphorylation at tyrosine residues also occurs, and has begun to attract a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Cantley et al., 1991, Cell 64:281-302; Hunter T., 1991, Cell 64:249-270; Nurse, 1990, Nature 344:503-508; Schlessinger et al., 1992, Neuron 9:383-391; Ullrich et al., 1990, Cell 61:203-212). Subversion of normal growth control pathways leading to oncogenesis has been shown to be caused by activation or overexpression of protein tyrosine kinases which constitute a large group of dominant oncogenic proteins (reviewed in Hunter, T., 1991, Cell 64:249-270).
2.2. Protein Tyrosine Kinases
Protein tyrosine kinases comprise a large family of proteins, including many growth factor receptors and potential oncogenes, which share ancestry with, but nonetheless differ from, serine/threonine-specific protein kinases (Hanks et al., 1988, Science 241:42-52). The protein kinases may further be defined as being receptors or non-receptors.
Receptor-type protein tyrosine kinases, which have a transmembrane topology have been studied extensively. The binding of a specific ligand to the extracellular domain of a receptor protein tyrosine kinase is thought to induce receptor dimerization and phosphorylation of their own tyrosine residues. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signalling molecules, thereby activating various signal transduction pathways (Ullrich et al., 1990, Cell 61:203-212).
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane domain. Within this broad classification, one can divide the known cytoplasmic protein tyrosine kinases into four distinct morphotypes: the SRC family (Martinez et al., 1987, Science 237:411-414; Sukegawa et al., 1987, Mol. Cell. Biol. 7:41-47; Yamanishi et al., 1987, 7:237-243; Marth et al., 1985, Cell 43:393-404; Dymecki et al., 1990, Science 247:332-336), the FES family (Ruebroek et al., 1985, EMBO J. 4:2897-2903; Hao et al., 1989, Mol. Cell. Biol. 9:1587-1593), the ABL family (Shtivelman et al., 1986, Cell 47:277-284; Kruh et al., 1986, Science 234:1545-1548), and the JAK family. While distinct in their overall molecular structure, each of the members of these morphotypic families of cytoplasmic protein tyrosine kinases share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains are the SH2 (SRC homology domain 2; Sadowski et al., Mol. Cell. Biol. 6: 4396-4408; Koch et al., 1991, Science 252:668-674) domains and SH3 domains (Mayer et al., 1988, Nature 332:269-272). Non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson et al., 1992, Cell 71:359-362).
While the metabolic roles of cytoplasmic protein tyrosine kinases are less well understood than that of the receptor-type protein tyrosine kinases, significant progress has been made in elucidating some of the processes in which this class of molecules is involved. For example, lck and fyn, members of the src family, have been shown to interact with CD4/CD8 and the T cell receptor complex, and are thus implicated in T cell activation, (Veillette et al., 1992, TIG 8:61-66). Certain cytoplasmic protein tyrosine kinases have been linked to certain phases of the cell cycle (Morgan et al., 1989, Cell 57:775-786; Kipreos et al., 1990, Science 248: 217-220; Weaver et al., 1991, Mol. Cell. Biol. 11:4415-4422). Cytoplasmic protein tyrosine kinases have been implicated in neuronal development (Maness, P., 1992, Dev. Neurosci. 14:257-270). Deregulation of kinase activity through mutation or overexpression is a well-established mechanism underlying cell transformation (Hunter et al., 1985, supra; Ullrich et al., supra).
2.3. G-Proteins and Signal Transduction
Guanine-nucleotide-binding proteins, (G-proteins; Simon et al., 1991, Science 252:802-808; Kaziro et al., 1991, Ann. Rev. Biochem. 60:349-400) such as Ras (for review, see Lowy et al., 1993, Ann Rev. Biochem. 62:851-891), play an essential role in the transmission of mitogenic signals from receptor tyrosine kinases. Taking Ras as an example, the activation of receptor tyrosine kinases by ligand binding results in the accumulation of the active GTP bound form of the Ras molecule (Gibbs et al., 1990, J. Biol. Chem. 265:20437-2044; Satoh et al., 1990, Proc. Natl. Acad. Sci. USA 87:5993-5997; Li et al., 1992, Science 256:1456-1459; Buday et al., 1993, Mol. Cell. Biol. 13:1903-1910; Medema et al., 1993, Mol. Cell. Biol. 13:155-162). Ras activation is also required for transformation by viral oncogenic tyrosine kinases (Smith et al., 1986, Nature 320:540-43).
Ras activity is regulated by the opposing actions of the GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, with GAPs stimulating the slow intrinsic rate of GTP hydrolysis on Ras and exchange factors stimulating the basal rate of exchange of GDP for GTP on Ras. Thus, GAPS act as negative regulators of Ras function, while exchange factors act as Ras activators.
Recently, a direct link between activated receptor tyrosine kinases and Ras was established with the finding that the mammalian GRB-2 protein, a 26 kilodalton protein comprised of a single SH2 and two SH3 domains (Lowenstein et al., 1992, Cell 70:431-442), directly couples receptor tyrosine kinases to the Ras exchange factor Sos in mammals and Drosophila (Buday et al., 1993, Cell 73:611-620; Egan et al., 1993, Nature 363:45-51; Li et al., 1993, Nature 363:85-87; Gale et al., 1993, Nature 363:88-92; Rozakis-Adcock et al., 1993, Nature 363:83-85; Chardin et al., 1993, Science 260:1338-1343; Oliver et al., Cell 73:179-191; Simon et al., 1993, Cell 73:169-177). The GRB-2 SH2 domain binds to specific tyrosine phosphorylated sequences in receptor tyrosine kinases while the GRB-2 SH3 domains bind to proline-rich sequences present in the Sos exchange factor. Binding of GRB-2 to the receptor kinases, therefore, allows for the recruitment of Sos to the plasma membrane, where Ras is located (Schlessinger, J., 1993, TIBS 8:273-275).
2.4. BCR-ABL in the Development of Leukemias
Activation of the oncogenic potential of normal cellular proteins such as protein tyrosine kinases may occur by alteration of the proteins"" corresponding enzymatic activities, their inappropriate binding to other cellular components, such as those mentioned above in Section 2.3, or both.
For example, the BCR-ABL protein tyrosine kinase oncoprotein may transform cells via changes in enzyme activity and/or altering of noncovalent protein-protein interactions. The gene encoding the BCR-ABL oncoprotein is a chimeric oncogene generated by the translocation of sequences from the cABL protein tyrosine kinase on chromosome 9 into BCR sequences on chromosome 22 (reviewed in Kurzock et al., 1988, N. Engl. J. Med. 319:990-998, and Rosenberg et al., 1988, Adv. in Virus Res. 35:39-81). The BCR-ABL oncogene has been implicated in the pathenogenesis of Philadelphia chromosome (Ph1) positive human leukemias. Namely, the Ph1 chromosome is found in at least 90 to 95 percent of cases of chronic myelogenous leukemia (CML), which is a clonal cancer arising from the neoplastic transformation of hematopoietic stem cells (Fialkow et al., 1977, Am. J. Med. 63:125-130), and is also observed in approximately 20 percent of adults with acute lymphocytic leukemia (ALL), 5 percent of children with ALL, and 2 percent of adults with acute myelogenous leukemia (AML) (Whang-Peng et al., 1970, Blood 36:448-457; Look, A. T., 1985, Semin. Oncol. 12:92-104). The BCR-ABL gene produces two alternative chimeric proteins, P210 BCR-ABL, and P185 BCR-ABL, which are characteristic of CML and ALL, respectively. Further, it has recently been directly demonstrated that the BCR-ABL gene product is the causative agent in CML (Skorski et al., 1993, J. Clin Invest. 92:194-202; Snyder et al., 1993, Blood 82:600-605).
Clinically, CML is characterized by a biphasic course. The disease begins with a chronic phase marked by a greatly increased pool of uncommitted myeloid progenitor cells. Because terminal differentiation is maintained, this results in greatly increased pools of circulating mature granulocytes. After a period of several weeks to many years, a state of accelerated myeloproliferation develops wherein the myeloid cells progressively lose their capacity for terminal differentiation. During this time, thrombocytosis, basophilia, and clonal cytogenetic abnormalities often appear. These abnormalities signal the terminal, blast-crisis stage, during which immature blast cells rapidly proliferate and the patient inevitably dies.
It has previously been shown that the BCR-ABL proteins exhibit heightened tyrosine kinase and transforming capabilities compared to the normal c-ABL protein (Konopka et al., 1984, Cell 37:1035-1042). BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL (Muller et al., 1991, Mol. Cell. Biol. 11:1785-1792; McWhirter et al., 1991, Mol. Cell. Biol. 11:1553-1565). The BCR first exon is capable of binding to the ABL SH2 domain in a phosphotyrosine-independent manner (Pendergast et al., 1991, Cell 66:161-171), and deletion of BCR sequences essential for ABL SH2-binding render BCR-ABL nontransforming (Pendergast et al., 1991, Cell 66:161-171). In addition, it has been demonstrated that BCR binds, in vitro, to some other SH2 domains encoded by other proteins (Muller et al., 1992, Mol. Cell. Biol. 12:5087-5093). While one may infer from these results that some aspect of SH2 domain-binding to BCR is involved in the oncogenicity of the BCR-ABL oncoprotein, the mechanism by which such BCR-ABL oncogenesis occurs is still obscure. For example, given the myriad of SH2-containing proteins which are known to exist, the identification of a BCR-ABL effector(s) will necessitate much further study.
The present invention relates to compositions and methods for the prevention and treatment of cell proliferative disorders wherein a protein tyrosine kinase or a protein tyrosine phosphatase capable of complexing with a member of the SH2- and/or SH3-containing family of adaptor proteins is involved. The present invention further relates to protein tyrosine kinase/adaptor protein complexes, protein tyrosine phosphatase/adaptor protein complexes, and the uses of these complexes for the identification of agents capable of disrupting the interaction between the components of such complexes.
xe2x80x9cProtein tyrosine kinasexe2x80x9d will, herein, be abbreviated xe2x80x9cPTKxe2x80x9d, and xe2x80x9cprotein tyrosine phosphatasexe2x80x9d will, herein be abbreviated xe2x80x9cPTPxe2x80x9d. It is to be understood that xe2x80x9cPTKxe2x80x9d may refer to either a transmembrane, receptor-type protein tyrosine kinase or a cytoplasmic protein tyrosine kinase, unless otherwise indicated, and, likewise, xe2x80x9cPTPxe2x80x9d may refer to either a transmembrane, receptor-type protein tyrosine phosphatase or a cytoplasmic protein tyrosine phosphatase, unless otherwise indicated.
This invention is based, in part, on the surprising discovery that the adaptor protein, GRB-2, binds the intracellular BCR-ABL PTK product in vivo and is necessary for the activation of the oncogenic potential of the BCR-ABL product. The inventors are the first to demonstrate the physiological relevance of the interactions between the members of a signal transduction pathway, in part by showing that disruption of this signal transduction can result in the reversal of the transformed phenotype of cells and inhibit tumor growth in animals. The data representing this discovery is presented in the Working Examples in Sections 6 through 12, below. The invention, therefore, represents the first instance whereby the SH2- and/or SH3-containing adaptor family of proteins, especially the GRB-2 member of the GRB subfamily of proteins, are implicated in the development and maintenance of cell proliferation/activationxe2x80x94herein demonstrated for the abnormal cellular proliferation involved in oncogenesis, the transformation process, and the development of human cancer. Still further, with respect to BCR-ABL transformation, the present invention discloses the first effector (i.e., GRB-2) for the BCR-ABL product.
3.1. Abbreviations
The following table lists the single-letter and triple-letter abbreviations for amino acids that are in common use among protein chemists and that are used herein.